Methods of degrading napalm B

ABSTRACT

Methods of degrading napalm and/or trinitrotoluene involve contacting the waste with specific intra-amoebic isolates of ATCC 40908 and/or dispersants derived therefrom. Useful isolates include is deposited as ATCC 77529, NAP-1 deposited as ATCC 77526 and 13 deposited as ATCC 77527.

The United States Government has rights in this invention pursuant tocontract No. DE-AC05-84OR21400 between the United States Department ofEnergy and Martin Marietta Energy Systems, Inc.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.08/011,841, filed on Feb. 1, 1993, now U.S. Pat. No. 5,314,821, issuedon May 24, 1994, entitled Amoeba/Bacteria Consortia and Uses forDegrading Wastes and Contaminants, the entire disclosure of which isincorporated herein by reference application Ser. No. 08/011,841 is acontinuation of application Ser. No. 07/693,998, filed on Apr. 26, 1991,now abandoned.

FIELD OF THE INVENTION

The present invention relates to methods of biological degradation ofnapalm and trinitrotoluene (TNT), and more particularly to such methodswhich utilize intra-amoebic isolates. Napalm, as used herein, is definedas Napalm B, a mxiture of gasoline, benzene, and polystyrene. Theoriginal, obsolete napalm formula, containing napthenic and palmiticacids, has not been tested.

BACKGROUND OF THE INVENTION

The end of the "cold war" has brought about the need for the reductionor elimination of many weapons stockpiles. Among those are explosivesand incendiaries which, for the sake of protecting the environment, canno longer be burned. Therefore there is a need for alternative methodsfor eliminating explosives and incendiaries, in particular, waste napalmand TNT.

OBJECTS OF THE INVENTION

Accordingly, it is an object of the present invention to provide a newand improved method of biological degradation of napalm.

It is another object of the present invention to provide a new andimproved method of biological degradation of TNT.

Further and other objects of the present invention will become apparentfrom the description contained herein.

SUMMARY OF THE INVENTION In accordance with one aspect of the invention,the foregoing and other objects are achieved by a method of degradingnapalm which involves the steps of:

a. providing a culture of a bacterium which comprises an intra-amoebicisolate essentially identical to American Type Culture CollectionDeposit Number 77529, a mutant thereof possessing all the identifyingcharacteristics thereof, or mixtures thereof;

b. deriving an aqueous dispersant solution from the culture;

c. contacting napalm B with a sufficient amount of the dispersantsolution to emulsify the napalm B; and,

d. contacting the emulsified napalm B with a sufficient amount of atleast one of intraamoebic isolates essentially identical to AmericanType Culture Collection Deposit Numbers 77526 and 77527, a mutantthereof possessing all the identifying characteristics thereof, ormixtures thereof, to degrade the emulsified napalm B.

DETAILED DESCRIPTION OF THE INVENTION

Amoeba/bacteria consortium 46, ATCC Deposit Reference No. 40908, wasfound to contain certain useful bacteria, designated as intra-amoebicisolates 13, NAP-1, 1S and CR-1. Isolate 1S produces a biodispersantwhich separates the components of napalm, and also elutes TNT bound tosoil. Isolates NAP-1 and 13, in combination with the biodispersantproduced from isolate 1S, degrade and disperse the polystyrene componentof napalm. Isolate CR-1 degrades TNT in a manner which renders itundetectable by standard analysis methods.

The method of deriving a dispersant and using it to separate componentsof napalm is described in general terms as follows: Isolate 1S isusually grown on culture plates containing a solid medium. A typicalsolid medium suitable for this purpose comprises 15g/L Bacto Tryptone(Pancreatic Digest of Casein), 5g/L Bacto Soytone (Papaic Digest ofSoybean Meal), 5g/L Sodium Chloride and 15g/L Bacto Agar. Small amountsof freshly grown bacteria (typically 1-3 day old cultures) are spread onthe surface of the solid medium and incubated at a temperature of about15° C. to about 40° C., preferably about 30° C., for several days(usually about 3-5 days) in an aerobic environment. When growth issufficiently heavy, the bacteria are harvested, washed by centrifugation(usually about three times) with normal saline (about 8.5g/L NaCl),diluted (to about 10 ml for each culture plate used) with normal saline,and autoclaved (typically for about 15 minutes at 121° C. at 15 psi).The autoclaved solution is allowed to cool, and is preferably filteredthrough a 0.2μm nucleopore filter to remove extraneous membranes andother bacterial debris. The biodispersant solution thus derived ispreferably stored in a sterile container. The biodispersant solution ispreferably diluted 1:10 with sterile distilled water for use inseparation of napalm components.

Napalm typically composed of polystyrene, leaded gasoline, and benzene,is degraded as follows: Napalm, preferably in its liquid form, is addedto the diluted biodispersant solution. The mixture should generallycontain about 5% to about 25%, preferably about 10%, napalm. Uponthorough mixing, an emulsifying effect is observed in the mixture,indicating suspension of the polystyrene component of the napalm.

To further degrade the mixture, a supernatant bacterial preparationcontaining about 10³ /ml to about 10⁸ /ml, usually about 10⁵ /ml,isolate 13 and/or isolate NAP-1, preferably both, is added to theemulsified mixture in a ratio of about 3:1 to about 1:3, preferablyabout 1:1. The mixture is then incubated at a temperature of about 15°C. to about 40° C., preferably about 30° C., until the polystyrenecomponent of the napalm has dissolved, usually at least 24 hours. Markedincreases in breakdown products of polystyrene, indicative of itsdegradation, will be evident, as seen in Table 1.

EXAMPLE

A dispersant was derived from Isolate 1S as described hereinabove, andwas mixed with napalm as described hereinabove to produce an emulsifiedmixture containing a suspension of the polystyrene component of thenapalm. Results are shown in Table 1.

EXAMPLE II

Isolate 13 and isolate NAP-1 were grown separately on Trypticase SoyAgar at 30° C. for 3 days and harvested dry. The bacteria were preparedand used as described hereinabove to degrade the mixture produced inExample I. Results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Effect of Isolates 13 and Nap-1 on Volatile Aromatic                          Components of Napalm                                                                   Water   Bio-      Biodispersant +                                             Control dispersant                                                                              Isolates 13, NAP-1                                 ______________________________________                                        Benzene in:                                                                   Polystyrene.sup.a                                                                        37,000,000                                                                              28,000,000                                                                              38,000,000                                     Supernate.sup.b                                                                          140,000   320,000   110,000                                        Ethyl Benzene in:                                                             Polystyrene                                                                              80,000    80,000    150,000                                        Supernate  420       620       150,000                                        Toluene in:                                                                   Polystyrene                                                                              81,000    91,000    130,000                                        Supernate  1,800     2,400     210,000                                        Total xylene in:                                                              Polystyrene                                                                              180,000   150,000   280,000                                        Supernate  1,200     820       260,000                                        ______________________________________                                         .sup.a μg/kg (semi solid portion of mixture)                               .sup.b μg/L (liquid portion of mixture)                               

A general, simple method of treating soil contaminated with TNT isdescribed as follows. Soil contaminated with TNT is loaded into avessel, usually a column, for contacting the soil with a biodispersant.An aqueous solution containing biodispersant derived from isolate 1 S(as described hereinabove) is added and allowed to percolate downthrough the soil and elute the TNT. The solution may be forced throughthe soil, the soil may be pretreated, and/or other process steps knownto the skilled artisan may be taken to increase the efficiency of theprocess. The process may be carried out at about room temperature, butprocess temperature is not a particularly critical factor.

EXAMPLE III

10 grams of contaminated soil were loaded into two columns, 7 in. inheight with a diameter of 2.0 inches, each column having a Whatman #1filter pad

in the bottom thereof to retain the soil. One column was eluted with 100ml

distilled, deionized water, and the other column was eluted with 100 mlof the above described dispersant solution. The flow rate through thesoil was slow, requiring 12 hours for all the liquid to pass through thecolumns. The eluate and soil for both the control and test columns werecollected and analyzed for TNT and metabolites after acetonitrileextraction. No metabolites were detected. The results are shown in Table2, indicating a significant reduction in detectable TNT in the soileluted with the biodispersant solution.

                  TABLE 2                                                         ______________________________________                                        Desorption of TNT from Soil Using Biodispersants from                         Isolate 1S                                                                             VA Site (soil)                                                                         NJ Site (soil)                                                                           NJ Site (eluate)                                 ______________________________________                                        Water Control                                                                            5,900 μg/g                                                                            370 μg/g                                                                              15,750 μg/L                               10% Biodispersant                                                                        1,900 μg/g                                                                             5.3 μg/g                                                                              1,100 μg/L                               ______________________________________                                    

A method of degrading TNT using isolate CR-1 is as follows. A solutionis prepared containing a mineral salts solution, usually NATE, and about20 mg/L to about 200 rag/L, usually about 50 mg/L, TNT. Isolate CR-1 isgrown on a suitable medium, usually Trypticase Soy Agar, for generally1-2 days at about 15° C. to about 40° C., usually about 30° C., andharvested dry. Isolate CR-1 is added to the TNT solution at aconcentration of about 10³ /ml to about 10⁹ /ml, usually about 106/ml,followed by incubation at about 15° C. to about 40° C., usually about30° C., to degrade the TNT.

A typical NATE solution is generally prepared using the followingmaterials:

Solution A (10X stock)

10 g/L MgSO₄. 7H₂ O

2 g/L CaCl₂

10 g/L KNO₃

1 g/L NH₄ Cl

Solution B (100X stock)

5 mg/L CuSO₄. 5H₂ O

1 mg/L H3BO₃

1 mg/L MnSO₄ or 0.76 mg/L MNSO₄. 1 H₂ O

7 mg/L ZnSO₄

1 mg/L MoO₃

1 mg/L CoCl₂. 6H₂ O

Phosphate Buffer

8.5 g KH₂ PO₄ and 6.5 g K₂ HPO₄ in 300 ml

Iron Chloride

0.027 g/100 ml FeCl₃

To prepare a typical NATE media, 100 ml of solution B is added to 1liter of solution A, resulting in a 10X NATE solution which can beautoclaved (sterilized). After cooling, the sterilized 10X NATE solutionis diluted 1:10 with sterile water to obtain a 1X NATE solution. To 1liter of the 1X NATE solution is added 20 ml filter sterilized phosphatebuffer and add 10 ml of filter sterilized FeCl₃ to obtain the finishedNATE media.

EXAMPLE IV

TNT was dissolved in water and diluted 1:1 with NATE media in a testbottle, resulting in a solution containing about 50 mg/L TNT. IsolateCR-1 was grown on Trypticase Soy Agar for 1-2 days at 30° C. and thenharvested dry. Isolate CR-1 was then added to the TNT/NATE solution at aconcentration of 10⁶ /ml, followed by incubation at 30° C. until thesolution had attained a stable yellow-orange color. A control for thisexperiment was similar to the test, with the organisms killed byautoclaving prior to addition to the test bottle. Each bottle wasanalyzed by high pressure liquid chromatography (HPLC) and showed amarked decrease in TNT levels by the live bacteria. Small quantities of4-amino-2,6-dinitrotoluene (4-ADNT), one of the metabolites associatedwith the degradation of TNT, were detected as indicated in Table 3.

                  TABLE 3                                                         ______________________________________                                        CONCENTRATION OF TNT AND ITS METABOLITES                                      AFTER INCUBATION WITH BACTERIA ISOLATE                                        CR-1                                                                          Isolate      TNT, mg/L  4-ADNT, mg/L                                          ______________________________________                                        CR-1         36         0                                                     (5 minutes)                                                                   CR-1         10         0.3                                                   (24 hours)                                                                    Control      48         0                                                     (24 hours)                                                                    ______________________________________                                    

EXAMPLE VI

Additional experiments were then undertaken using a similar protocol aspreviously mentioned except that the saturated TNT solution nowcontained ¹⁴ C-labeled TNT. Most of the ⁴ C-TNT was associated with thecell pellet. Further analysis of the cell pellet showed no detectableTNT present. TNT metabolites were detected in small amounts in the testbottles. Results are shown in Tables 4 and 6.

                  TABLE 4                                                         ______________________________________                                        % .sup.14 C-TNT per fraction                                                  EXPERIMENT   Cell Pellet  CO.sub.2                                                                             Soluble                                      ______________________________________                                        1            70           1      29                                           2            66           2      32                                           Control       6           4      90                                           ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        % TNT and Metabolites in Cell Pellet                                          EXPERIMENT   TNT       2-ADNT   4-ADNT                                        ______________________________________                                        1            0.0       0.009    0.04                                          2            0.0       0.005    0.05                                          Control      0.0       0.000    0.00                                          ______________________________________                                    

Deposit of Microorganisms

The applicants, in accordance with the provisions of the Budapest treatyon the international recognition of the deposit of microorganisms forthe purposes of patent procedure under the Budapest Treaty, did depositsamples of Isolate NAP-1, Isolate 13, Isolate CR-1, and Isolate 1S withthe American Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, U.S.A. on Aug. 20, 1993 and assigned ATCC depositreference Numbers 77526, 77527, 77528, and 77529, respectively. Eachculture is hereby irrevocably and without restriction or conditionreleased to the public upon the issuance of letters patent herefor.

While there has been shown and described what are at present consideredthe preferred embodiments of the invention, it will be obvious to thoseskilled in the art that various changes and modifications can be madetherein without departing from the scope of the inventions defined bythe appended claims.

What is claimed is:
 1. A method of degrading napalm B comprising thesteps of:a. providing a culture of a bacterium comprising anintra-amoebic isolate possessing all the identifying characteristics ofAmerican Type Culture Collection Deposit Number 77529, a mutant of saidisolate possessing all the identifying characteristics thereof, ormixtures thereof; b. deriving an aqueous dispersant solution from saidculture; c. contacting napalm B with a sufficient amount of saiddispersant solution to emulsify said napalm B producing emulsifiednapalm B; and, d. contacting said emulsified napalm B with a sufficientamount of at least one other intra-amoebic isolate possessing all theidentifying characteristics of American Type Culture Collection DepositNumbers 77526, and ATCC No. 77527, a mutant thereof possessing all theidentifying characteristics thereof, or mixtures thereof, to degradesaid emulsified napalm B; and, e. degrading said emulsified napalm B. 2.A method according to claim 1 wherein said dispersant solution isdiluted 1:10 with water after step b, and wherein, in step c, saidnapalm B is added to said diluted dispersant solution in an amount inthe range of about 5% to about 20%.
 3. A method according to claim 2wherein said amount of napalm B is about 10%.
 4. A method according toclaim 1 wherein said other intra-amoebic isolate is a supernatantbacterial preparation containing about 10₃ /ml to about 10₈ /ml of saidother isolate.
 5. A method according to claim 4 wherein said supernatantbacterial preparation contains about 10⁵ /ml of said other isolate.
 6. Amethod according to claim 1 wherein, in step d, said other intra-amoebicisolate is added to said emulsified napalm B in a ratio in the range ofabout 3:1 to about 1:3.
 7. A method according to claim 6 wherein saidratio is about 1:1.
 8. A method according to claim 1 wherein step d iscarried out at a temperature in the range of about 15° C. to about 40°C.
 9. A method according to claim 16 wherein said temperature is about30° C.